The capacity to produce high levels of alcohol is a very rare characteristic in nature. It is most prominent in the yeast Saccharomyces cerevisiae, which is able to accumulate in the absence of cell proliferation, ethanol concentrations in the medium of more than 17%, a level that kills virtually all competing microorganisms. As a result, this property allows this yeast to outcompete all other microorganisms in environments rich enough in sugar to sustain the production of such high ethanol levels (Casey and Ingledew, 1986; D'Amore and Stewart, 1987). Very few other microorganisms, e.g., the yeast Dekkera bruxellensis, have independently evolved a similar but less pronounced ethanol tolerance compared to S. cerevisiae (Rozpedowska et al., 2011). The capacity to accumulate high ethanol levels lie at the basis of the production of nearly all alcoholic beverages as well as bioethanol in industrial fermentations by the yeast S. cerevisiae. Originally, all alcoholic beverages were produced with spontaneous fermentations in which S. cerevisiae gradually increases in abundance, in parallel with the increase in the ethanol level, to finally dominate the fermentation at the end.
The genetic basis of yeast alcohol tolerance, particularly ethanol tolerance has attracted much attention but until recently nearly all research was performed with laboratory yeast strains, which display much lower alcohol tolerance than the natural and industrial yeast strains. This research has pointed to properties like membrane lipid composition, chaperone protein expression and trehalose content, as major requirements for ethanol tolerance of laboratory strains (D'Amore and Stewart, 1987; Ding et al., 2009) but the role played by these factors in other genetic backgrounds and in establishing tolerance to very high ethanol levels has remained unknown. We have recently performed polygenic analysis of the high ethanol tolerance of a Brazilian bioethanol production strain VR1. This revealed the involvement of several genes previously never connected to ethanol tolerance and did not identify genes affecting properties classically considered to be required for ethanol tolerance in lab strains (Swinnen et al., 2012a).
A second shortcoming of most previous studies is the assessment of alcohol tolerance solely by measuring growth on nutrient plates in the presence of increasing alcohol levels. (D'Amore and Stewart, 1987; Ding et al., 2009). This is a convenient assay, which allows hundreds of strains or segregants to be phenotyped simultaneously with little work and manpower. However, the real physiological and ecological relevance of alcohol tolerance in S. cerevisiae is its capacity to accumulate by fermentation high alcohol levels in the absence of cell proliferation. This generally happens in an environment with a large excess of sugar compared to other essential nutrients. As a result, a large part of the alcohol in a typical, natural or industrial, yeast fermentation is produced with stationary phase cells in the absence of any cell proliferation. The alcohol tolerance of the yeast under such conditions determines its maximal alcohol accumulation capacity, a specific property of high ecological and industrial importance. In industrial fermentations, a higher maximal alcohol accumulation capacity allows a better attenuation of the residual sugar and, therefore, results in a higher yield. A higher final alcohol titer reduces the distillation costs and also lowers the liquid volumes in the factory, which has multiple beneficial effects on costs of heating, cooling, pumping and transport of liquid residue. It also lowers microbial contamination and the higher alcohol tolerance of the yeast generally also enhances the rate of fermentation especially in the later stages of the fermentation process. Maximal alcohol accumulation capacity can only be determined in individual yeast fermentations, which are much more laborious to perform than growth tests on plates. In static industrial fermentations, maintenance of the yeast in suspension is due to the strong CO2 bubbling and this can only be mimicked in lab scale with a sufficient amount of cells in a sufficiently large volume.
The advent of high-throughput methods for genome sequencing has created a breakthrough also in the field of quantitative or complex trait analysis in yeast (Liti and Lewis, 2012; Swinnen et al., 2012b). The new methodology has allowed efficient QTL mapping of several complex traits (Swinnen et al., 2012a; Ehrenreich et al., 2010; Parts et al., 2011) and reciprocal hemizygosity analysis (Steinmetz et al., 2002) has facilitated identification of the causative genes. The efficiency of the new methodologies calls for new challenges to be addressed, such as comparison of the genetic basis of related complex properties. In addition, complex trait analysis in yeast has been applied up to now mainly to phenotypic properties that are easy to score in hundreds or even thousands of segregants (Swinnen et al., 2012a; Ehrenreich et al., 2010; Parts et al., 2011; Steinmetz et al., 2002; Winzeler et al., 1998; Deutschbauer and Davis, 2005; Brem et al., 2002; Marullo et al., 2007; Nogami et al., 2007; Perlstein et al., 2007). However, many phenotypic traits with high ecological or industrial relevance require more elaborate experimental protocols for assessment and it is not fully clear yet whether the low numbers of segregants that can be scored in these cases are adequate for genetic mapping with pooled-segregant whole-genome sequence analysis.